Molecular Mechanism of Polarized Growth in Pollen Tubes

花粉管极化生长的分子机制

• 批准号: 9724047
• 负责人: Zhenbiao Yang
• 金额: $ 28.5万
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 1999-10-19
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9724047&HistoricalAwards=false
• 关键词: Molecular; Mechanism; Polarized; Growth; Pollen

9724047 Yang Tip-growing pollen tubes grow directionally toward the ovules to delivery sperms to the embryo sac and thus are critical for sexual reproduction in higher plants. Pollen tubes also present an attractive model system for the study of polarized growth, partly because they are haploid and can be easily cultured. The long-term goal of this project is to elucidate molecular pathways that govern polarized cell growth in pollen tubes. We have identified a rho-type small GTP-binding protein, Rop1, that preferentially expressed in pollen and pollen tubes and is localized to the apex of pea and Arabidopsis pollen tubes. Antibody microinjection experiments indicate that Rop1 plays a pivotal role in pollen tube growth likely by modulating Ca2+-dependent exocytosis. The goal of this proposed work is to confirm the role of Rop1 in polarized tube growth using a reverse genetics approach and to investigate underlying molecular and cellular mechanisms for the function of Rop1 in pollen tubes. The role of Rop1 will be genetically tested using transgenic Arabidopsis plants that express dominant positive and negative mutant Rop1 alleles under the control of a pollen tube-specific promoter. The transgenic pollen tubes will be analyzed for changes in tip growth and in vivo guidance. The transgenic pollen tubes expressing Rop1 dominant mutant alleles will also be analyzed for changes in the behavior of secretory vesicles using TEM and the rapid freeze and freeze substitution technique and for changes in the influx of extracellular Ca2+ and the tip-focused intracellular Ca2+-gradients using a Ca2+-selective vibrating probe and ratiometric ion imaging. These careful analyses will test the notion that Rop1 controls polarized cell growth by modulating Ca2+ signaling-dependent polarized exocytosis. To further understand the underlying mechanism for Rop1-mediated polarized growth, Rop1 effectors will be identified by cloning genes encoding Rop1 partners using interactive cloning techniques such as the yeast two-hybrid method. Rop1 effectors are expected to interact specifically with GTP-bound Rop1 (dominant positive mutant) but not with GDP-bound Rop1 (dominant negative mutant). These studies will establish a solid foundation for our long-term goal of elucidating molecular pathways that lead to directional pollen tube growth. This work may also generate a broad impact on understanding the molecular basis of polarized growth in higher organisms as well as produce useful information for manipulating sexual reproduction in higher plants, such as engineering male sterility for hybrid production. The polar growth of pollen tubes is an interesting and significant process in which a germinating pollen tube extends in a single direction to mediate fertilization and reproduction in plants. The PI has identified a protein (ROP1, GTP-binding protein) involved in the signal transduction system that appears to control the polar growth of pollen tubes through calcium fluxes. With this preliminary data as a base the PI will use mutants of ROP1 to examine the function of the protein. Using light microscopy assays the role of ROP1 in inducing a calcium fluxes will be visualized and correlated with electron microscopy of the intracellular events that are required to extend the pollen tube. ROP1 must interact with other proteins in cascade that transmits signals through the cell. Proteins that interact with ROP1 will be identified and characterized. This research is of fundamental importance in describing a critical process in the reproduction of plants. Practical applications of the research include modifying plant reproductive growth to create superior plants. ***

9724047阳尖的花粉管向胚珠方向生长，以将精子递送到胚胎囊中，因此对于高等植物的性繁殖至关重要。 花粉管还为研究极化生长提供了一个有吸引力的模型系统，部分原因是它们是单倍体的，并且很容易培养。 该项目的长期目标是阐明控制花粉管中极化细胞生长的分子途径。 我们已经确定了一种小型GTP结合蛋白ROP1，该蛋白在花粉和花粉管中优先表达，并位于豌豆和拟南芥花粉管的顶点。 抗体显微注射实验表明，ROP1在花粉管生长中起关键作用，可能通过调节Ca2+依赖性胞吐作用。 这项提出的工作的目的是使用反向遗传学方法来确认ROP1在极化管生长中的作用，并研究ROP1在花粉管中ROP1功能的基本分子和细胞机制。 ROP1的作用将使用转基因拟南芥植物进行遗传测试，这些植物在花粉管特异性启动子的控制下表达占主导地位的正和阴性突变型ROP1等位基因。 将分析转基因花粉管的尖端生长和体内指导的变化。 还将分析表达ROP1显性突变等位基因的转基因花粉管，使用TEM和快速冻结和冷冻替代技术的分泌囊泡行为发生变化，以及使用CA2+-SELECTY PROCTICECTECTICTIPRICTIPECECIEN和RAYONTIC IIN和RAYOMENTICIONTION和RAYOMENTIONT和RAYONTICIONTION。 这些仔细的分析将测试ROP1通过调节CA2+信号依赖性极化胞吐作用来控制极化细胞生长的观点。 为了进一步了解ROP1介导的极化生长的潜在机制，将通过使用交互式克隆技术（例如酵母双杂交方法）编码ROP1伙伴的克隆基因来识别ROP1效应子。 ROP1效应子有望与与GTP结合的ROP1（主要阳性突变体）特别相互作用，但与GDP结合的ROP1（主要负突变体）不相互作用。 这些研究将为我们阐明导致方向花粉管生长的分子途径的长期目标建立坚实的基础。 这项工作还可能对理解高等生物的两极化生长的分子基础产生广泛的影响，并为操纵高等植物的性繁殖提供有用的信息，例如工程男性无菌性用于混合生产。 花粉管的极性生长是一个有趣而重要的过程，其中发芽的花粉管沿单个方向延伸以介导植物的施肥和繁殖。 PI鉴定了信号转导系统中涉及的蛋白质（ROP1，GTP结合蛋白），该蛋白似乎通过钙通量控制花粉管的极性生长。使用此初步数据作为基础，PI将使用ROP1的突变体检查蛋白质的功能。使用光显微镜测定，ROP1在诱导钙通量中的作用将被可视化并与延伸花粉管所需的细胞内事件的电子显微镜相关。 ROP1必须与级联中的其他蛋白质相互作用，该蛋白质通过细胞传输信号。与ROP1相互作用的蛋白质将被识别和表征。这项研究对于描述植物繁殖的关键过程至关重要。该研究的实际应用包括修改植物生殖生长以创造出色的植物。 ***